Cell cycle regulation is a very complicated and delicate process, containing cell growth, differentiation, proliferation and apoptosis. In the past decades, the discovery in process of cell cycle signal transduction pathway is a great milestone in molecular biology history. Protein kinases regulate many critical biological mechanisms, including metabolism and cell growth, proliferation, and differentiation. Aberrations in the activity of the kinases involved in signal transduction have been linked to many human diseases such as cancer, diabetes and inflammation. More than 600 kinases have been encoded on human genome The cyclin-dependent kinases (Cdks) are a prominent family of protein kinases, which plays a key role in cell-cycle progression and cellular proliferation. Cdks are a serine/threonine protein kinase, multi-subunit enzymes composed of at least a catalytic subunit protein kinases and a regulatory subunit cyclin. Cdks exert their effective activation of host proteins through phosphorylation of key serine or threonine residues by ATP. The phosphorylated proteins modulate the activity of a variety of cellular proteins. Mitosis is activated by the driver of Cdk1/Cyclin B, which has been widely accepted. It has been found that 90% of tumor formation is related to the highly activated Cdks led by Rb tumor suppressor protein phosphorylation, which result Rb-path deactivation. Cdks inhibitor is effective in inducing apoptosis and improving the sensitivity of chemotherapy drugs. Cdks have become attractive therapeutic targets for anti-cancer drug discovery. [Science, 2004, 303, 1800; The AAPS Journal, 2006, 8, E204; Cancer Biology Therapy, 2003, 4, S84]
Natural flavonoids are found in many plants with various biological activities, and are one series of natural Cdks inhibitors. However, natural flavonoids are often poor water soluble, with low bioavailability and Cdks inhibitory activity. In order to enhance potency, structure modification of natural flavonoids is of great importance and value.
In 1994, through globally natural anti-cancer drug screening by U.S. National Cancer Institute (NCI), Rohitukine, which is an alkaloid flavonoid from Indian plant Dysoxylum binectariferum, was selected and exhibited moderate anti-cancer activity. Flavopiridol, which is a derivative derived from rohitukine, demonstrated potent Cdks inhibitory activity and low toxicity. Flavopiridol is firstly synthesized by Aventis Corporation, a new amino synthetic derivative of natural flavonoid. Flavopiridol was initially discovered as an anti-proliferative epidermal growth factor receptor kinase inhibitor. Subsequent studies show, however, that Flavopiridol is an even more potent inhibitor of Cdks. In addition, it is also proved to be an mRNA transcription inhibitor. Flavopiridol has been the first anti-cancer drug candidate which targets cell cycle signal transduction, and currently has been in the process of Phase II clinical trial in U.S.A. Iowa State University researchers have found that Flavopiridol may hinder the AIDS virus HIV-1 in the cell proliferation, and significantly increase the sensitivity of chemotherapy drugs. P-TEFb has also been proved to be highly inhibited by Flavopiridol in relative low dose, which also indicates promising prospect in anti-cancer and anti-AIDS therapy. [Cancer Biology Therapy, 2003, 4, S84, 77, 146; Curr. Pharm. Des., 2006, 12, 1949] An interesting finding was that apoptosis inhibitor survivin was Cdk1/Cyclin B phosphorylation, may cancel its anti-apoptotic activity, where as survivin expresses only in malignant tumors, not in mature tissues. Flavopiridol can reduce the concentration of survivin in malignant tumor cells indirectly, through inhibiting Cdk1/Cyclin B. Louis Staudt from NCI revealed that the mechanism of Flavopiridol inducing cancer cell apoptosis and HIV proliferation inhibition was attributed to the blockage of transcription mRNA of most gene, and only targeted anti-cancer cells selectively by the use of DNA chip technology. This is because RNA in the immoderate growing cancer cells is very short-lived, which is totally dependent on the incessant synthesis of mRNA. Once Flavopiridol enter into the cells, the synthesis of mRNA is blocked. Short-lived RNA in cancer cells are degradated rapidly, while the majority of normal cells with long-lived mRNA still exist, so it only induce cancer cells apoptosis selectively. Flavopiridol is a selective Cdks inhibitor interacting specifically with the ATP binding site of Cdks. It inhibits growth of cancer cells through inducting apoptosis, unlike cytotoxic anti-cancer drugs which kill cells directly. It means that although Cdks exists in all series of human cells, cancer cells would be inhibited to grow mostly for their considerable multiplication compared to normal cells. So, less harm would be against the normal human cells. [Genome Biology, 2001, 2 (10), 0041.1] Flavopiridol can also improve the sensitivity of chemotherapy drugs effectively, and has obvious synergies effect and suppression of “multi-drug resistance”, which can also be used in advanced staged cancer treatment so as to improve life quality and lengthen life time. However, Flavopiridol is a chiral compound with complex, low yielded and high-cost synthesis processes. Moreover, there is still a severe side effect in its clinical application, such as diarrhea and so on. In a word, the discovery of competitive Cdks-ATP binding site inhibitor Flavopiridol has brought new treatment channel for cancer and AIDS.
In 2008, P276-00, like Flavopiridol, is a novel Cdks inhibitor of organic amine derivatives of flavonoid, currently in phase II clinical trials as a potential anti-cancer agent by Kalpan S. Joshi. [WO2004/004632] However, unlike Flavopiridol, it shows better selectivity toward Cdk1-B, Cdk4-D1, and Cdk9-T1 as compared with Cdk7-H and Cdk2-E. The inhibition to 12 kinds of human tumor cells growth in culture is twice to thrice higher than Flavopiridol, while less toxic than Flavopiridol and almost has no inhibitory activity on two human embryonic normal lung fibroblast WI-38 and MRC-5. However, similar to Flavopiridol, poor solubility and low bioavailability of P276-00 may affect its effects of Cdks inhibitors. [Molecular Cancer Therapeutics, 2007, 6, 918]
Scutellaria baicalensis Georgi is a kind of the most widely used traditional herbal medicines and plants the largest of cultivated varieties of medicinal herbs in China. As the main active ingredient, Scutellaria flavonoids is as high as 10-20%, with the function of anticancer, antimicrobial, anti-inflammatory, anti-allergic, anti-oxidation, anti-blood fat, anti-thrombosis and so on. As a natural Cdks inhibitor, Baicalein and Wogonin are stronger than Scutellaria flavonoid glycosides. Baicalein and Wogonin can induce apoptosis, inhibit cell proliferation, and inhibit HIV replication and reverse transcriptase. The function of anti-tumor and anti-AIDS and so on suggests that there is a unique role, has been extensive attention. [Cancer Treatment Reviews, 2009, 35, 57] However, skullcap flavonoids are insoluble in water, easily oxidized and low serum concentration of active strength, so they do not reach clinical needs. Therefore, the inventors hope to find new efficient ATP-selective inhibitor of Cdks with low toxicity through structural modification of skullcap flavonoids. The flavonoid derivatives U.S. Pat. No. 5,849,733 disclosed Flavopiridol of thio- and oxo-derivatives as Cdks inhibitors for the treatment of proliferative diseases. CN16686131A (WO2004/004632) disclosed flavonoid derivatives as Cdks inhibitors. U.S. Pat. No. 5,116,954 disclosed with the anti-tumor and immunomodulatory activity of flavonoids. CN1990481 disclosed a kind of baicalein derivatives, their preparation methods and uses. CN1427003A disclosed inhibit the protein kinase C activity of 8-substituted methylamine baicalein derivatives and their preparation methods. The patent did not mention the present invention involved in skullcap flavonoid organic amine derivatives, as well as Cdks inhibitors purposes.